Understanding the selective vulnerability of different neurons in the context of Alzheimer's Disease (AD) may hold the key to developing an urgently needed, effective therapy for this devastating disease. We propose that selective vulnerability is controlled by the balance of protective and sensitizing factors, which different neurons express at different levels. While transcriptomics and proteomics can catalog differences in gene expression between vulnerable and resilient neurons, the list of differentially expressed genes is long and fails to establish causality. Therefore, a functional approach is needed that enables precise control of the neuronal expression of all genes to establish their causal roles in AD vulnerability and their potential as therapeutic targets. Here, we propose to pioneer an innovative, multidisciplinary approach that integrates CRISPR-based genome-wide functional screening in human iPSC-derived neurons with single-cell expression analysis of human neurons exhibiting a gradient of vulnerability to AD and quantitative multiplex immunostaining of human AD brains at progressive AD neuropathological stages to uncover the factors controlling selective vulnerability in AD. MPI Dr. Kampmann has co-developed a CRISPR-based platform for repression (CRISPRi) and activation (CRISPRa) of gene expression in genome-wide functional screens and has implemented them in human iPSC- derived neurons. MPI Dr. Grinberg has a long track record in AD research and has pioneered several innovative approaches proposed here. As studies with iPSC-derived neurons and postmortem human brains offer different strengths, investigating of cellular factors in these complementary systems will serve as cross- validation enhancing the overall impact of this proposal. Our focus in this proposal is the accumulation of pathological species of the protein tau, which strongly correlates with neuronal death and cognitive decline in AD. We will conduct genome-wide CRISPRi and CRISPRa screens to identify factors controlling formation of pathological tau, and cellular stress hypersensitivity downstream of pathological tau in iPSC-derived cholinergic neurons (a proxy of early vulnerable basal forebrain neurons), and glutamatergic neurons (a proxy of moderately vulnerable cortical neurons). We will validate a role for the identified factors in post-mortem brains spanning all stages of AD progression. We will also identify differentially expressed genes in vulnerable and resilient neurons from AD brains by laser-microdissection coupled with RNA-Seq. We will test a functional role for differentially expressed genes in iPSC-derived neuron models. MPIs Dr. Kampmann and Dr. Grinberg, both located in the UCSF Neurosciences Center, are already closely collaborating in the context of the NINDS- funded U54 Center without Walls for Tau, in which Dr. Kampmann directs the CRISPRi/a Core and Dr. Grinberg directs the Human Biology Validation Core. We expect that the proposed research will reveal key factors controlling selective vulnerability, and thereby potential therapeutic targets.